Pathogens express their virulence genes in response to specific signals detected during infection. The PhoP/PhoQ and PmrA/PmrB systems are key regulators of Salmonella pathogenicity. We have now determined that activation of these two systems by their respective signals is dependent on the Salmonella-specific regulatory gene ssrB. This application describes experiments to examine: first, the mechanism by which SsrB enables the PhoP/PhoQ system to respond to mildly acidic pH; second, how the SsrB protein enables normal activation of the PmrA/PmrB system by ferric iron; and third, how the activities of the virulence control systems PhoP/PhoQ, PmrA/PmrB, OmpR/EnvZ and SsrB/SpiR change over time when Salmonella is inside macrophages and in response to mildly acidic pHs. These four regulatory systems share the ability to respond to mildly acidic pH, to regulate expression of other systems and to be highly active inside macrophages. By achieving these goals, we shall gain an understanding of how pathogens perceive signals denoting a host environment and how they can expand their sensing abilities by acquiring new genes. The proposed research will reveal the changing activities of key virulence regulatory systems when a pathogen experiences the dynamic host cell milieu.